Infectious diseases remain the largest cause of death in the world today, greater than cardiovascular disease or cancer..sup.1 Among infectious diseases, tuberculosis (TB) is the leading cause of death..sup.2 The emergence of multidrug-resistant (MDR) strains and the global human immunodeficiency virus (HIV) pandemic amplify the incidence of TB.
Tuberculosis mainly affects the lungs but can also involve other organs. TB strikes people of all ages but is more common among the elderly. The disease can also afflict animals, especially livestock such as cattle, hogs, and poultry. Rod-shaped bacteria, tubercle bacilli discovered by the German physician Robert Koch in 1882, cause the disease. Tubercle bacilli belong to a genus of bacteria called Mycobacterium. This disease once ranked among the most common causes of death in the world. Today, improved methods of prevention, detection, diagnosis, and treatment have greatly reduced both the number of people who contract the disease and the number of people who die from it. However, in the last decade, the outbreaks of MDR tuberculosis (MDRTB) and TB amplified by the global HIV pandemic make TB an urgent global issue.
One-third of the world's population is infected with Mycobacterium tuberculosis (Mtb),.sup.3 a facultative intracellular bacillus. After infection with Mtb, the lifetime risk of developing TB is approximately 10%, while 90% of infected persons have latent infection with viable bacilli. This 10% rate of TB accounts for the 8 million persons reported annually with active TB, and the resultant 3 million deaths. Moreover, TB is a serious problem faced by hemodialysis patients,.sup.4 and TB is the No. 1 killer of women of childbearing age around the world with 1.2 million women dying of the disease in 1997 according to reports by the World Health Organization..sup.11a
a. Tuberculosis and AIDS
TB infection is a serious problem for acquired immunodeficiency syndrome (AIDS) patients. HIV-infected individuals are particularly susceptible to infection with Mtb and the development of TB. Compared to an individual who is not infected with HIV, an individual infected with HIV has a 10 times greater risk of developing TB. In an individual infected with HWV, the presence of other infections, including TB, may allow HIV to multiply more quickly. This may result in more rapid progression of HIV infection and AIDS.5 As HIV infection progresses, CD4+ lymphocytes decline in number and function. The immune system is less able to prevent the growth and local spread of Mtb. Even in HIV-infected patients, pulmonary TB (PTB) is still the most common form of TB. The presentation depends on the degree of immunosuppression.
As in adults, the natural history of TB in a child infected with HIV depends on the stage of HIV disease. Early in HIV infection, when immunity is strong, the signs of TB are similar to those in a child without HIV infection. As HIV infection progresses and immunity declines, dissemination of TB becomes more commnon. Tuberculous meningitis, miliary tuberculosis, and widespread tuberculous lymphadenopathy occur.
HIV-positive patients and staff in health units face daily exposure to TB. The risk of exposure is greatest in adult medical wards and TB wards where there are many PTB cases. From 1990-1992, the Centers for Disease Control and prevention (CDC) investigated outbreaks of multidrug-resistant tuberculosis (MDRTB) in several hospitals and a state correctional system. Almost 300 cases of MDRTB were identified in these outbreaks; most patients were HIV-seropositive. The mortality rate was 80%-90% and the median interval from diagnosis of tuberculosis to death ranged from 4-16 weeks..sup.6 In 1995, about one third of the 17 million HIVnfected people worldwide were also co-infected with Mtb..sup.5 (TB is the leading cause of death in AIDS patients).
b. Treatment of TB
Isoniazid (isonicotinic acid hydrazide) (INH) was first reported to be effective against Mtb and M. bovis in 1952..sup.7-9 Isoniazid, now still a front-line therapy against TB, has been shown to be an effective prophylactic antitubercular.sup.10, and modern short-course chemotherapy is initiated with three drugs: isoniazid, rifampin and pyrazinamide (PZA), often with the inclusion of a fourth drug, usually ethambutol. Recently, rifapentine, a derivative of rifamycin, was approved by the FDA for the treatment of tuberculosis. .sup.11b
The American Thoracic Society and the CDC in the United States now recommend a treatment regimen of isoniazid, rifampin, and pyrazinamide for 2 months, followed by isoniazid and rifampin for an additional 4 months, as the standard 6-month regime. Isoniazid, cheap and safe, has a wide therapeutic margin and high early bactericidal activity so that it kills rapidly growing bacilli in lesions, but is inefficient in ultimately sterilizing these lesions. Rifampin and PZA are crucial in achieving sterilization by killing persisting semi-dormant bacilli, and are thus responsible for shortening the duration of treatment from the earlier norm of 12-18 months to the current standard of 6 months..sup.12 However, many people fail to complete the lengthy therapy, treatment failures are high, and MDR is increasing. A 4-year study, led by the World Health Organization, shows that of people who had been treated for TB for less than a month, 36 percent harbored microbes that resisted at least one of the four main anti-tuberculosis drugs. Moreover, 10% of infected people who had never been treated for the disease carried a strain of Mtb that resisted at least one drug..sup.13
d. Mechanism of Drug Action
Isoniazid is a prodrug that requires activation by the mycobacterial catalase-peroxidase enzyme (KatG) to an active form that then exerts a lethal effect on an intracellular target or targets..sup.14-16 The lethal effect lies in the biosynthetic pathway for mycolic acids,.sup.14, 17-19 alpha-branched and beta-hydroxylated fatty acids found in the envelope of mycobacteria.
Rifamycins (e.g. rifampin, rifabutin and rifapentine) are potent inhibitors of prokaryotic DNA-dependent RNA polymerase,.sup.20 with little activity against the equivalent mammalian enzymes. This group of antimicrobial agents are compounds composed of aromatic rings linked by an aliphatic bridge. Most likely, the lipophilic properties of the molecule are important for the binding of the drug to the polymerase and aid in the penetration of the drug across the mycobacterial cell wall.
Pyrazinamide (PZA) is a synthetic derivative (pyrazine analog) of nicotinamide and in combination with isoniazid is rapidly bactericidal for replicating forms of Mtb, with an average MIC of 20 .mu.g/mL. The activity of PZA depends on the presence of a bacterial amidase which converts PZA to pyrazinoid acid (PZOA), the active antibacterial molecule..sup.21 Amidase activity is present in PZA-sensitive but not in PZA-resistant species such as M. bovis, opportunistic mycobacteria and Mtb resistant to PZA as a result of drug therapy. The gene (pncA) encoding the PZA (and nicotinamide) amidase which is responsible for processing PZA into its bactericidal form has been identified, and the mutations in pncA that confer PZA resistance to tubercle bacilli have been recently reported..sup.22
Ethambutol is active against Mtb, with MICs in the range of 1 to 5 .mu.g/mL. The drug has much more variable activity against the other species of slowly growing mycobacteria and is significantly less active against rapidly growing mycobacteria. On the whole, ethambutol is inactive against other microorganisms. The mechanisms of action of ethambutol have focused on two targets: polyamine function and metabolism and cell wall synthesis. Ethambutol inhibits the transfer of mycolic acid into the cell wall and stimulates trehalose dimycolate synthesis..sup.23
e. Multidrug Resistance
The importance of KatG mutations in isoniazid resistance is well established, although the extent to which such mutations account for the spectrum of resistance observed in clinical isolates is arguable..sup.24 Best estimates indicate that &gt;50% of isoniazid-resistant clinical isolates are KatG mutants..sup.25
Mycobacteria have a similar enzyme, InhA, required for mycolic acid biosynthesis..sup.26 A genetic approach revealed that InhA appeared to finction as a component of a type II fatty acid synthase system responsible for the final reduction step in chain elongation to form conventional fatty acids..sup.27-28 Sequencing of clinical isolates of Mtb has revealed mutations in putative regulatory regions upstream of the InhA gene and potential coding sequence mutations that may be directly involved in isoniazid resistance, but these occur only in a subpopulation of isoniazid-resistant, wild-type catalase-peroxidase isolates..sup.24-25, 29-31 Thus, although the InhA protein may be involved in isoniazid-resistance, it probably does not represent the target whose inhibition results in hexacosanoic acid accumulation, and mutations in InhA and KatG do not appear to be sufficient to account for all of the observed resistance..sup.32 Recently, a protein species purified from INH-treated Mtb was shown to consist of a covalent complex of isoniazid, 12-kilodalton acyl carrier protein (AcpM), and a beta-ketoacyl acyl carrier protein synthase, KasA. Amino acid-altering mutations in the KasA protein were identified in isoniazid-resistant patient isolates that lacked other mutations associated with resistance to this drug..sup.33
Most recently, the complete genome sequence of the best-characterized strain of Mtb, H37Rv, has been determined and analyzed..sup.34 This will improve our understanding of the biology of this slow-growing pathogen and to help in the conception of new prophylactic and therapeutic interventions.
f. The Significance of the Recent TB Revival
Worldwide, the recorded number of new cases of TB correlates with economic conditions, the highest incidence being seen in Africa, Asia, and Latin America. In the industrialized nations, including Europe, the steady drop in TB incidence began to level off in the mid-1980s and then stagnated or even reversed somewhat. Much of this rise can be attributed to an influx of migrants from countries with a higher incidence of TB..sup.35
Another element in this rising trend is HIV. The particular susceptibility and increased mortality of the disease among individuals infected with HIV pose a serious threat to TB control programs..sup.36 Moreover, the emergence of multidrug-resistant strains of M. tuberculosis (MDR-Mtb) has resulted in fatal outbreaks in many countries, including the United States..sup.37 Strains of MDR-Mtb, some of which are resistant to as many as seven drugs, are deadly to both HIV negative and HIV positive individuals..sup.38 The occurrence of MDRTB in patients with AIDS has led to significant changes in the management of tuberculosis in these patients, compared with tuberculosis in patients without AIDS.
g. Prophylactic Treatment
The prophylactic treatment of children with strongly positive tuberculin test by isoniazid and rifampicin has brought a marked reduction in the incidence of pediatric TB in high incidence geographic areas..sup.39 Controlled clinical studies have shown that isoniazid preventive treatment reduces the risk of TB disease in HIV-positive individuals also infected with Mtb..sup.6 Since (+)-calanolide A might be used in the clinic as an anti-AIDS drug in the near future, it might demonstrate a prophylactic and therapeutic effect on TB for AIDS patients when used in AIDS treatment.